The long-term goals of my research program include defining the biochemical, molecular and endocrine aspects of RA (retinoic acid) biosynthesis. This project will focus on the second step in the pathway of RA biogenesis from retinol (vitamin A), catalyzed by RALDH1-4 (retinal dehydrogenase types 1-4), and will test the umbrella hypothesis that each RALDH makes a unique and essential contribution to RA biogenesis. Specific Aims 1 would determine the contribution of RALDH 1, 2 and 3 to RA biosynthesis in the brain, focusing on the hippocampus. This aim would test the hypotheses that: hippocampus neurons biosynthesize RA from retinol;BDNF enhances neuron conversion of retinol into RA;BDNF functions by increasing RALDH3 mRNA and protein;and exogenous retinoids alter RA biosynthesis in the brain. This aim will include quantifying RA concentrations in specific brain areas by LC/MS/MS (lower limit of detection of 10 fmol) during aging and treatment with exogenous retinoids. Specific Aims 2-4 will: 2) generate and evaluate RALDH4-null mice;3) identify the RALDH 1 gene regulatory regions that mediate tissue-specific responses to retinoids in vivo;4) generate and evaluate conditional RALDH2 knockouts. Each of these aims represents a logical progression in evaluating the specific contribution of an RALDH or RALDHs to RA biosynthesis. Each aim will rely on integrating molecular genetics approaches (transgenics and knockouts) with analytical techniques (hplc, LC/MS) and applying both to metabolic studies in vitro and in the whole animal. The fundamental insight and reagents generated will be useful to identify diseases and age-related degeneration that may be caused or exacerbated by impaired RA biosynthesis.